CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence

Abstract

Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

miR-3174 Is a New Tumor Suppressor MicroRNA That Inhibits Several Tumor-Promoting Genes in Glioblastoma

“…Dysregulation of CD44 is associated with multiple cancers including lung cancer [35], breast cancer [36], osteosarcoma [37] colorectal cancer [38] and glioma [39] etc. Moreover, a recent report has shown that depletion of CD44 in GBM cells induces senescence and inhibits growth and stemness [40].…”

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma

“…5). The AC-like 1 and Intermediate 1 cell states were predicted to receive intercellular signals most broadly across myeloid subtypes with receptors including FGFR1, IGF1R, and CD44, which is specifically known to interact with a range of ligands (e.g., HBEGF, PSEN1, SPP1, VEGFA) and has a critical role in adult glioma [34][35][36] . In contrast, the AClike 2 and Intermediate 2 populations were the most inert neoplastic populations (Figure 4g).…”

The Application of CRISPR-Cas System in Glioblastoma Research and Treatment

“…Similarly, a team from Uppsala University used CRISPR-Cas9 to knock out CD44 in the U251M cell line. CD44-/glioblastoma cells showed reduction of growth and stemness, and induction of senescence (Kolliopoulos, 2022). Cancer-specific insertion-deletions attacker (CINDELA) system designed by the University of British Columbia can specifically target tumor cells (Kwon, 2022).…”